Immunohistochemical and Confocal Imaging Protocol

Tissue samples embedded in paraffin blocks were obtained from the Biological Resource Centre of the Pathology Department, Centre Antoine Lacassagne, Nice. Informed written consent was obtained from all the patients in accordance with institutional guidelines and the experimental protocol, that complied with ethical and safety practices for research involving human tissues, was approved by the CPP Sud-Méditerranée V ethics committee (National Authorisation Number: AC-2014-2237). Immuno-staining was performed as described^{40 (link)}. For fluorescence, slides were labelled with appropriate secondary antibodies (Invitrogen) followed by nuclear staining with DRAQ5 (Thermo Scientific) and then mounted in ProLong Gold antifade reagent (Invitrogen). Immunohistochemically stained sections were digitalized using a Hamamatsu NanoZoomer 2.0-HT Digital slide scanner (40X mode). Images were visualized and captured using NDP.view2:U12388-01 software. Fluorescently-stained sections were analysed and images acquired with a 10x/0.45 or 40x/1.1 W objective on a Zeiss NLO780 confocal system.